T cells mediate most forms of cellular immunity, including cell lympholysis, delayed type hypersensitivity (DTH), transplantation rejection, and allograft rejection. An introduction to T cells and cell mediated immunity is found in Paul (1993) Fundamental Immunology, Third Edition Raven Press, New York, N.Y. and the references cited therein.
Typical T cells do not respond to free antigenic peptides. Instead, T cells interact with a specialized set of cell surface proteins (the class I and class II major histocompatibility complexes, or MHC) which present antigens on the surface of cells. Cytotoxic T cells are induced to proliferate by specialized antigen presenting cells such as macrophage and dendritic cells which present antigenic peptides on their cellular surfaces in conjunction with MHC molecules. T cells are induced by these antigen presenting cells to recognize corresponding antigens expressed on MHC antigens on the surface of target cells. T cells destroy these target cells.
The T cell recognizes the antigen in the form of a polypeptide fragment bound to the MHC class I molecules on target cells, rather than the intact polypeptide itself. The polypeptide is endogenously synthesized by the cell, and a portion of the polypeptide is degraded into small peptide fragments in the cytoplasm. Some of these small peptides translocate into a pre-Golgi compartment and interact with class I heavy chains to facilitate proper folding and association with the subunit β2 microglobulin. The peptide-MHC class I complex is then routed to the cell surface for expression and potential recognition by specific T cells. Investigations of the crystal structure of the human MHC class I molecule HLA-A2.1 indicate that a peptide binding groove is created by the folding of the α1 and α2 domains of the class I heavy chain (Bjorkman et al., (1987) Nature 329:506. Falk et al., (1991) Nature 351:290 have developed an approach to characterize naturally processed peptides bound to class I molecules. Other investigators have successfully achieved direct amino acid sequencing of the more abundant antigenic peptides in various HPLC fractions by conventional automated sequencing of peptides eluted from class I molecules (Jardetzky, et al. (1991) Nature 353:326 and mass spectrometry Hunt, et al., Science 225:1261 (1992). A review of the characterization of naturally processed peptides in MHC Class I is found in Rötzschke and Falk (1991) Immunol. Today 12:447.
Target T cells recognizing antigenic peptides can be induced to differentiate and proliferate in response to antigen presenting cells bearing antigenic peptides in the context of MHC class I and class II complexes. There are differences in the antigenic peptides bound to MHC class I and class II molecules, but the two classes of bound peptides share common epitopes within the same protein which enable a T cell activated by an antigen presenting cell to recognize a corresponding MHC class I epitope. MHC class I molecules on target cells typically bind 9 amino acid antigenic peptides, while corresponding MHC class II-peptide complexes have greater heterogeneity in the size of the bound antigenic peptide.
The generation of target T cells with a desired specificity has been limited by the ability of investigators to discover appropriate peptides for loading onto MHC molecules, and by investigator's ability to load peptide antigens onto antigen presenting cells used to induce proliferation of the T cells. In the past, investigators have generated antigen presenting cells by stripping the antigenic peptides normally found on antigen presenting cells by chemical or thermal techniques, followed by a reloading of the cells with a desired antigenic peptide. This approach has had limited success, due to inefficiencies in antigen presenting cell peptide loading, and due to the limited length of time that the loaded antigenic peptides remain loaded on the antigen presenting cells. In addition, only a single peptide fragment of a protein is loaded onto the surface of the antigen presenting cell using typical methods; thus, peptides important for activation of T cells against a target cell can be overlooked. The present invention overcomes these and other problems.